Isogenic Mammary Models of Intraductal Carcinoma Reveal Progression to Invasiveness in the Absence of a Non-Obligatory In Situ Stage.

In breast cancer, progression to invasive ductal carcinoma (IDC) involves interactions between immune, myoepithelial, and tumor cells. Development of IDC can proceed through ductal carcinoma in situ (DCIS), a non-obligate, non-invasive stage, or IDC can develop without evidence of DCIS and these cases associate with poorer prognosis. Tractable, immune-competent mouse models are needed to help delineate distinct mechanisms of local tumor cell invasion and prognostic implications. To address these gaps, we delivered murine mammary carcinoma cell lines directly into the main mammary lactiferous duct of immune-competent mice. Using two strains of immune-competent mice (BALB/c, C57BL/6), one immune-compromised (severe combined immunodeficiency; SCID) C57BL/6 strain, and six different murine mammary cancer cell lines (D2.OR, D2A1, 4T1, EMT6, EO771, Py230), we found early loss of ductal myoepithelial cell differentiation markers p63, α-smooth muscle actin, and calponin, and rapid formation of IDC in the absence of DCIS. Rapid IDC formation also occurred in the absence of adaptive immunity. Combined, these studies demonstrate that loss of myoepithelial barrier function does not require an intact immune system, and suggest that these isogenic murine models may prove a useful tool to study IDC in the absence of a non-obligatory DCIS stage-an under-investigated subset of poor prognostic human breast cancer.

The Protective Effects of Flavonoids in Cataract Formation through the Activation of Nrf2 and the Inhibition of MMP-9.

Cataracts account for over half of global blindness. Cataracts formations occur mainly due to aging and to the direct insults of oxidative stress and inflammation to the eye lens. The nuclear factor-erythroid-2-related factor 2 (Nrf2), a transcriptional factor for cell cytoprotection, is known as the master regulator of redox homeostasis. Nrf2 regulates nearly 600 genes involved in cellular protection against contributing factors of oxidative stress, including aging, disease, and inflammation. Nrf2 was reported to disrupt the oxidative stress that activates Nuclear factor-κB (NFκB) and proinflammatory cytokines. One of these cytokines is matrix metalloproteinase 9 (MMP-9), which participates in the decomposition of lens epithelial cells (LECs) extracellular matrix and has been correlated with cataract development. Thus, during inflammatory processes, MMP production may be attenuated by the Nrf2 pathway or by the Nrf2 inhibition of NFκB pathway activation. Moreover, plant-based polyphenols have garnered attention due to their presumed safety and efficacy, nutritional, and antioxidant effects. Polyphenol compounds can activate Nrf2 and inhibit MMP-9. Therefore, this review focuses on discussing Nrf2's role in oxidative stress and cataract formation, epigenetic effect in Nrf2 activity, and the association between Nrf2 and MMP-9 in cataract development. Moreover, we describe the protective role of flavonoids in cataract formation, targeting Nrf2 activation and MMP-9 synthesis inhibition as potential molecular targets in preventing cataracts.

Myocardial infarction classification and its implications on measures of cardiovascular outcomes, quality, and racial/ethnic disparities.

Heart disease continues to be the leading cause of death in the United States, with approximately 805 000 cumulative deaths from myocardial infarctions (MI) from 2005 to 2014. Gender and racial/ethnic disparities in MI diagnoses are becoming more evident in quality review audits. Although recent changes in diagnostic codes provided an improved framework, clinically distinguishing types of MI remains a challenge. MI misdiagnoses and health disparities contribute to adverse outcomes in cardiac medicine. We conducted a literature review of relevant biomedical sources related to the classification of MI and disparities in cardiovascular care and outcomes. From the studies analyzed, African Americans and women have higher rates of mortality from MI, are more probably to be younger and present with other comorbidities and are less probably to receive novel therapies with respect to type of MI. As high-sensitivity troponin assays are adopted in the United States, implementation should account for how race and sex differences have been demonstrated in the reference range and diagnostic threshold of the newer assays. More research is needed to assess how the complexity of health disparities contributes to adverse cardiovascular outcomes. Creating dedicated medical quality teams (physicians, nurses, clinical documentation improvement specialists, and medical coders) and incorporating a plan-do-check-adjust quality improvement model are strategies that could potentially help better define and diagnose MI, reduce financial burdens due to MI misdiagnoses, reduce cardiovascular-related health disparities, and ultimately improve and save lives.

Myoepithelial cell differentiation markers in ductal carcinoma in situ progression.

We describe a preclinical model that investigates progression of early-stage ductal carcinoma in situ (DCIS) and report that compromised myoepithelial cell differentiation occurs before transition to invasive disease. Human breast cancer MCF10DCIS.com cells were delivered into the mouse mammary teat by intraductal injection in the absence of surgical manipulations and accompanying wound-healing confounders. DCIS-like lesions developed throughout the mammary ducts with full representation of human DCIS histologic patterns. Tumor cells were incorporated into the normal mammary epithelium, developed ductal intraepithelial neoplasia and DCIS, and progressed to invasive carcinoma, suggesting the model provides a rigorous approach to study early stages of breast cancer progression. Mammary glands were evaluated for myoepithelium integrity with immunohistochemical assays. Progressive loss of the myoepithelial cell differentiation markers p63, calponin, and α-smooth muscle actin was observed in the mouse myoepithelium surrounding DCIS-involved ducts. p63 loss was an early indicator, calponin loss intermediate, and α-smooth muscle actin a later indicator of compromised myoepithelium. Loss of myoepithelial calponin was specifically associated with gain of the basal marker p63 in adjacent tumor cells. In single time point biopsies obtained from 16 women diagnosed with pure DCIS, a similar loss in myoepithelial cell markers was observed. These results suggest that further research is warranted into the role of myoepithelial cell p63 and calponin expression on DCIS progression to invasive disease.

Adipophilin regulates maturation of cytoplasmic lipid droplets and alveolae in differentiating mammary glands.

Milk lipids originate by secretion of triglyceride-rich cytoplasmic lipid droplets (CLDs) from mammary epithelial cells. Adipophilin (ADPH)/Plin2, a member of the perilipin family of CLD binding proteins, is hypothesized to regulate CLD production in these cells during differentiation of the mammary gland into a secretory organ. We tested this hypothesis by comparing CLD accumulation in differentiating mammary glands of wild-type and ADPH-deficient mice. ADPH deficiency did not prevent CLD formation; however, it disrupted the increase in CLD size that normally occurs in differentiating mammary epithelial cells. Failure to form large CLDs in ADPH-deficient mice correlated with localization of adipose triglyceride lipase (ATGL) to the CLD surface, suggesting that ADPH promotes CLD growth by inhibiting lipolytic activity. Significantly, mammary alveoli also failed to mature in ADPH-deficient mice, and pups born to these mice failed to survive. The possibility that CLD accumulation and alveolar maturation defects in ADPH-deficient mice are functionally related was tested by in vivo rescue experiments. Transduction of mammary glands of pregnant ADPH-deficient mice with adenovirus encoding ADPH as an N-terminal GFP fusion protein prevented ATGL from localizing to CLDs and rescued CLD size and alveolar maturation defects. Collectively, these data provide direct in vivo evidence that ADPH inhibition of ATGL-dependent lipolysis is required for normal CLD accumulation and alveolar maturation during mammary gland differentiation. We speculate that impairing CLD accumulation interferes with alveolar maturation and lactation by disrupting triglyceride homeostasis in mammary epithelial cells.

Prolactin-mediated regulation of lipid biosynthesis genes in vivo in the lactating mammary epithelial cell.

Prolactin (PRL) is known to play an essential role in mammary alveolar proliferation in the pregnant mouse, but its role in lactation has been more difficult to define. Genetic manipulations that alter expression of the PRL receptor and its downstream signaling molecules resulted in developmental defects that may directly or indirectly impact secretory activation and lactation. To examine the in vivo role of PRL specifically in lactation, bromocriptine (BrCr) was administered every 8 h to lactating mice on the second day postpartum, resulting in an ~95% decrease in serum PRL levels. Although morphological changes in secretory alveoli were slight, by 8 h of BrCr, pup growth was inhibited significantly. Phosphorylated STAT5 fell to undetectable levels within 4 h. Decreased milk protein gene expression, ß-casein, and α-lactalbumin, was observed after 8 h of treatment. To assess mammary-specific effects on lipid synthesis genes, we isolated mammary epithelial cells (MECs) depleted of mammary adipocytes. Expression of genes involved in glucose uptake, glycolysis, pentose phosphate shunt, de novo synthesis of fatty acids, and biosynthesis of triacylglycerides was decreased up to 19-fold in MECs by just 8 h of BrCr treatment. Glands from BrCr-treated mice showed a twofold reduction in intracellular cytoplasmic lipid droplets and a reduction in cytosolic ß-casein. These data demonstrate that PRL signaling regulates MEC-specific lipogenic gene expression and that PRL signals coordinate the milk synthesis and mammary epithelial cell survival during lactation in the mouse.

The adipophilin C terminus is a self-folding membrane-binding domain that is important for milk lipid secretion.

Cytoplasmic lipid droplets (CLD) in mammary epithelial cells undergo secretion by a unique membrane envelopment process to produce milk lipids. Adipophilin (ADPH/Plin2), a member of the perilipin/PAT family of lipid droplet-associated proteins, is hypothesized to mediate CLD secretion through interactions with apical plasma membrane elements. We found that the secretion of CLD coated by truncated ADPH lacking the C-terminal region encoding a putative four-helix bundle structure was impaired relative to that of CLD coated by full-length ADPH. We used homology modeling and analyses of the solution and membrane binding properties of purified recombinant ADPH C terminus to understand how this region possibly mediates CLD secretion. Homology modeling supports the concept that the ADPH C terminus forms a four-helix bundle motif and suggests that this structure can form stable membrane bilayer interactions. Circular dichroism and protease mapping studies confirmed that the ADPH C terminus is an independently folding α-helical structure that is relatively resistant to urea denaturation. Liposome binding studies showed that the purified C terminus binds to phospholipid membranes through electrostatic dependent interactions, and cell culture studies documented that it localizes to the plasma membrane. Collectively, these data provide direct evidence that the ADPH C terminus forms a stable membrane binding helical structure that is important for CLD secretion. We speculate that interactions between the four-helix bundle of ADPH and membrane phospholipids may be an initial step in milk lipid secretion.

Multiple functions encoded by the N-terminal PAT domain of adipophilin.

Adipophilin (ADPH), a member of the perilipin family of cytoplasmic lipid droplet (CLD)-binding proteins, is crucially dependent on triglyceride synthesis for stability. We have used cell lines expressing full-length or N-terminally modified forms of ADPH to investigate the role of the N-terminus in regulating ADPH stability and interactions with CLD. Full-length ADPH was unstable and could not be detected on CLDs unless cultures were incubated with oleic acid (OA) to stimulate triglyceride synthesis, or were treated with MG132 to block proteasomal degradation. By contrast, ADPH lacking amino acids 1-89 (Delta 2,3 ADPH), or N-terminally GFP-tagged full-length ADPH, was stable in the absence of OA or MG132, as was the closely related protein TIP47. However, none of these proteins localized to CLDs unless OA was added to the culture medium. Furthermore, immunofluorescence analysis showed that TIP47 localization to CLDs was prevented by full-length ADPH, but not by Delta 2,3 ADPH. These results suggest that the N-terminal region of ADPH mediates proteasomal degradation and access of TIP47 to the CLD surface and possibly contributes to CLD stability. Chimeras of ADPH and TIP47, generated by swapping their N- and C-terminal halves, showed that these properties are specific to ADPH.

Mammary glands of adipophilin-null mice produce an amino-terminally truncated form of adipophilin that mediates milk lipid droplet formation and secretion.

Adipophilin (ADPH), a member of the perilipin family of lipid droplet-associated proteins, is hypothesized to mediate milk lipid formation and secretion. Unexpectedly, the fat content of milk from ADPH-null mice was only modestly lower than that of wild-type controls, and neither TIP47 nor perilipin appeared to fully compensate for ADPH loss. This prompted us to investigate the possibility that the mutated ADPH gene was not a genuine null mutation. ADPH transcripts were detected in ADPH-null mammary tissue by quantitative real-time PCR, and C-terminal-specific, but not N-terminal-specific, ADPH antibodies detected a single lower molecular weight product and immunostained cytoplasmic lipid droplets (CLDs) and secreted milk fat globules in ADPH-null mammary tissue. Furthermore, stable cell lines expressing cDNA constructs corresponding to the ADPH-null mutation produced a product comparable in size to the one detected in ADPH-null mammary glands and localized to CLDs. Based on these data, we conclude that ADPH-null mice express an N-terminally truncated form of ADPH that retains the ability to promote the formation and secretion of milk lipids.

Molecular determinants of milk lipid secretion.

Mammary epithelial cells secrete lipids by an envelopment process that produces lipid droplets coated by membranes derived from the plasma membrane and possibly secretory vesicles. This secretion process, which resembles viral budding, is hypothesized to be mediated by specific interactions between molecules on the surface of intracellular lipids and membrane elements of the cell. Multiple lines of evidence indicate that milk lipid secretion occurs through a tripartite complex between the integral transmembrane protein, butyrophilin (BTN); the soluble metabolic enzyme, xanthine oxidoreductase (XOR); and the lipid droplet surface protein, adipophilin (ADPH). However, topological evidence from freeze-fracture replica immunolabelling (FRIL) challenge this model and suggests that milk lipid secretion is mediated by butyrophilin alone. Advances in our understanding of the molecular, structural, and functional properties of these proteins now make it possible to understand the physiological functions of each of these molecules in detail and to identify the specific molecular determinants that mediate milk lipid secretion.

Impaired tight junction sealing and precocious involution in mammary glands of PKN1 transgenic mice.

The mammary gland undergoes a complex set of changes to establish copious milk secretion at parturition. To test the hypothesis that signaling through the Rho pathway plays a role in secretory activation, transgenic mice expressing a constitutively activated form of the Rho effector protein PKN1 in the mammary epithelium were generated. PKN1 activation had no effect in late pregnancy but inhibited milk secretion after parturition, diminishing the ability of transgenic dams to support a litter. Mammary gland morphology as well as increased apoptosis and expression of IFGBP5 and TGFbeta3 suggest precocious involution in these animals. Furthermore, tight junction sealing at parturition was impaired in transgenic mammary glands as demonstrated by intraductal injection of [14C]sucrose. Consistent with this finding, tight junction sealing in response to glucocorticoid stimulation was highly impaired in EpH4 mammary epithelial cells expressing constitutively activated PKN1, whereas expression of a dominant-negative PKN1 mutant resulted in accelerated tight junction sealing in vitro. Tight junction formation was not impaired as demonstrated by the correct localization of occludin and ZO1 at the apical cell borders. Our results provide evidence that PKN1 participates in the regulation of tight junction sealing in the mammary gland by interfering with glucocorticoid signaling.

Cytoplasmic lipid droplet accumulation in developing mammary epithelial cells: roles of adipophilin and lipid metabolism.

PAT proteins (perilipin, adipophilin, and TIP47) are hypothesized to be critical regulators of lipid accumulation in eukaryotic cells. We investigated the developmental relationships between the expression of these proteins and cytoplasmic lipid droplet (CLD) accumulation in differentiating secretory epithelial cells in mouse mammary glands. Adipophilin (ADPH) specifically localized to CLD in differentiating and lactating mammary glands and was found exclusively in the secreted lipid droplet fraction of mouse milk. ADPH transcripts were selectively detected in secretory epithelial cells, and steady-state levels of both ADPH mRNA and protein increased during secretory differentiation in patterns consistent with functional linkage to CLD accumulation. TIP47 also was detected in secretory epithelial cells; however, it had a diffuse punctate appearance, and its mRNA and protein expression patterns did not correlate with CLD accumulation. Perilipin-positive adipose cells and steady-state levels of perilipin mRNA and protein decreased during mammary gland differentiation, suggesting a progressive loss of adipose lipid storage during this process. Collectively, these data demonstrate that increased ADPH expression is a specialized property of differentiated secretory epithelial cells and provide developmental evidence specifically linking increased ADPH expression to increased CLD accumulation. In addition, evidence is presented that the epithelial and adipose compartments of the mammary gland undergo concerted, developmentally regulated shifts in lipid metabolism that increase the availability of fatty acids necessary for lipid synthesis by milk-secreting cells.

Transduction of the mammary epithelium with adenovirus vectors in vivo.

Because the mammary parenchyma is accessible from the exterior of an animal through the mammary duct, adenovirus transduction holds promise for the short-term delivery of genes to the mammary epithelium for both research and therapeutic purposes. To optimize the procedure and evaluate its efficacy, an adenovirus vector (human adenovirus type 5) encoding a green fluorescent protein (GFP) reporter and deleted of E1 and E3 was injected intraductally into the mouse mammary gland. We evaluated induction of inflammation (by intraductal injection of [(14)C]sucrose and histological examination), efficiency of transduction, and maintenance of normal function in transduced cells. We found that transduction of the total epithelium in the proximal portion of the third mammary gland varied from 7% to 25% at a dose of 2 x 10(6) PFU of adenovirus injected into day 17 pregnant mice. Transduction was maintained for at least 7 days with minimal inflammatory response; however, significant mastitis was observed 12 days after transduction. Adenovirus transduction could also be used in the virgin animal with little mastitis 3 days after transduction. Transduced mammary epithelial cells maintained normal morphology and function. Our results demonstrate that intraductal injection of adenovirus vectors provides a versatile and noninvasive method of investigating genes of interest in mouse mammary epithelial cells.